Diversity, Cloning And Expression Of Protease Genes And Analysis Of Protease Of Deep Sea Strain Bacillus Sp. EPPRO6

A strain Bacillus sp. EPPRO6, which produced proteases, was isolated from the Pacific Ocean sediment samples. The cells of the strain were Gram-negative, in a single order and like short rods, with a size of 1.6～1.8μm×2.5～3.0μm and no flagella in electron micrograph. The strain could utilize various carbon and nitrogen sources, and produce proteases. Especially after induction of casein in the medium, the strain could produce a large number of intracellular proteases. The purification of crude enzyme was performed by ammonium sulfate fractionation and column chromatography with Sephadex G-75 and a purified enzyme with a molecular mass of 34 kDa was obtained after analysis of electrophoresis. Analysis of properties of purified protease showed that the optimum condition for the protease activity was 60℃and pH 8～9, which suggested that the enzyme was a typical alkaline mesothermal protease, with no sensibility to high temperature and high thermal stability. After incubation of 60℃for 1 hours, the protease still remained 50% activity. Ion Ca2+ could greatly enhance the thermal stability of the protease. The protease was stimulated by ions Co2+, Mg2+, Ca2+ etc. and inhibited by Fe3+,Hg2+,Cu2+ etc. Metal chelator EDTA with concentration of 10 mmol/L, and serine protease inhibitors PMSF and AEBSF could inhibited the protease, and cysteine protease inhibitor E-64 had no effects on the protease. The protease had some tolerance to urea and Mercaptoethanol.After amplification by PCR, 8 relevant protease gene fragments were gained. With the method of chromosome walking, totally 5 complete related protease genes (open reading frame) were obtained. Analysis of sequences and conserved regions showed that they were intracellular serine protease, peptidase M4, CAAX amino terminal protease, neutral protease B and microbial collagenase. The primers of expression for 5 complete protease genes were designed. Recombinant plasmids were transformed into E.coli BL21, in which three protease genes were expressed, and results of electrophoresis showed that all expressed proteins were in accordance with the expected sizes. Only expressed protein of serine protease gene was soluble and had the nature of serine protease. The experiment of traditional UV mutagenesis of wild-type strain was carried. It was expected to get a mutant strain which could produce protease that could tolerate much more acid, alkaline or have higher stability. The strain could produced various proteases with high activity, and excellent thermal stability which had a potential industrial application value.